Recently the field of reverse genetic analysis, or gene silencing, has been revolutionized by the discovery of potent, sequence specific inactivation of gene function, which can be induced by double-stranded RNA (dsRNA). This mechanism of gene silencing is termed RNA interference (RNAi), and it has become a powerful and widely used tool for the analysis of gene function in invertebrates and plants (reviewed in Sharp, P. A. (2001) Genes Dev 15, 485-90). Introduction of double-stranded RNA (dsRNA) into the cells of these organisms leads to the sequence-specific destruction of endogenous RNAs, when one of the strands of the dsRNA corresponds to or is complementary to an endogenous RNA. The result is inhibition of the expression of the endogenous RNA. Endogenous RNA can thus be targeted for inhibition, by selecting dsRNA of which one strand is complementary to the sense strand of an endogenous RNA. During RNAi, long dsRNA molecules are processed into 19-23 nucleotide (nt) RNAs known as short-interfering RNAs (siRNAs) that serve as guides for enzymatic cleavage of complementary RNAs (Elbashir, S. M. et al. (2001) Genes Dev 15, 188-2000; Parrish, S. et al. (2000) Mol Cell 6, 1077-87; Nykanen, A. et al. (2001) Cell 107, 309-21; Elbashir, S. M. et al. (2001) Embo J 20, 6877-88; Hammond, S. M. et al. (2000) Nature 404, 293-6; Zamore, P. D. et al. (2000) Cell 101, 25-33; Bass, B. L. (2001) Nature 411,428-9; and Yang, D. et al. (2000) Curr Biol 10, 1191-200). In addition, siRNAs can function as primers for an RNA-dependent RNA polymerase, leading to the synthesis of additional dsRNA, which in turn is processed into siRNAs to amplify the effects of the original siRNAs (Sijen, T. et al. (2001) Cell 107, 465-76; and Lipardi, C. et al. (2001) Cell 107, 297-307). Although the overall process of siRNA inhibition has been characterized, the specific enzymes that mediate siRNA function remain to be identified.
In mammalian cells, dsRNA is processed into siRNAs (Elbashir, S. M. et al. (2001) Nature 411, 494-8; Billy, E. et al. (2001) Proc Natl Acad Sci USA 98, 14428-33; and Yang, S. et al. (2001) Mol Cell Biol 21, 7807-16), but RNAi was not successful in most cell types due to nonspecific responses elicited by dsRNA molecules longer than about 30 nt (Robertson, H. D. & Mathews, M. B. (1996) Biochimie 78, 909-14). However, Tuschl and coworkers recently made the remarkable observation that transfection of synthetic 21-nt siRNA duplexes into mammalian cells effectively inhibits endogenous genes in a sequence specific manner (Elbashir, S. M. et al. (2001) Nature 411, 494-8; and Harborth, J. et al. (2001) J Cell Sci 114, 4557-65). These siRNA duplexes are too short to trigger the nonspecific dsRNA responses, but they still trigger destruction of complementary RNA sequences (Hutvagner, G. et al. (2001) Science 293, 834-8). This was a stunning discovery, and was followed by its utilization by several laboratories to knock out different genes in mammalian cells. The reported results demonstrate that siRNA appears to work quite well in most instances. However, a major limitation to the use of siRNA in host cells, and in particular in mammalian cells, is the method of delivery.
Currently, the synthesis of the siRNA is expensive. Moreover, inducing cells to take up exogenous nucleic acids is a short-term treatment and is very difficult to achieve in some cultured cell types. This methodology does not permit long-term expression of the siRNA in cells or use of siRNA in tissues, organs, and whole organisms. It had also not been demonstrated that siRNA could effectively be expressed from recombinant DNA constructs to suppress expression of a target gene. Thus, what is needed is more economical methods of synthesizing siRNAs. What is also needed are compositions and methods to express and deliver siRNA intracellularly in mammalian cells, and indeed in other cells as well. Such compositions and methods would have great utility not only as research tools, but also as a potent therapy for both infectious agents and for genetic diseases, by inhibiting expression of targeted genes.